Human cancers arise through a series of genetic changes that transform normal cells into malignant tumors. Many of these changes are caused by genomic rearrangements and other errors during replication. To prevent and repair such replication errors, cells have evolved DNA damage checkpoints, a sophisticated set of DNA quality control mechanisms. Central among them is the S-phase DNA damage checkpoint, a mechanism that slows replication in response to DNA damage. This checkpoint has two branches: one that regulates the activation of replication origins, and one that requires the MRN (Mre11-Rad50-Nbs1) recombination complex. The mechanism of the MRN-dependent branch is unknown. However, genetic evidence in humans and mice suggest that this branch of the S-phase DNA damage checkpoint is crucial for preventing cancer;human patients with mutations in Mre11 or Nbs1 are prone to a variety of early-onset malignancies. Understanding the mechanism of this checkpoint is essential for understanding the etiology of these cancers and will fundamentally affect the way subsequent studies of this checkpoint are approached. The proposed experiments are designed to test the hypothesis that the MRN-dependent branch acts to induce replication-coupled recombination. This hypothesis will be directly tested by quantitating the rate of recombination induced by DNA damage during replication and by determining if, and how, this recombination is regulated by the checkpoint. Complementary biochemical and genetic analysis of the MRN proteins will identify their specific mechanistic roles in the checkpoint, and will provide tools for studying the general checkpoint mechanism. These experiments will take advantage of the fission yeast Schizosaccharomyces pombe as a model system. The conservation of checkpoints between fission yeast and humans makes fission yeast an excellent model for investigating these vital DNA damage surveillance pathways. The powerful genetic and biochemical tools available for fission yeast make it possible to rapidly identify key pathway members and rigorously test hypotheses about their functions. Understanding the fission yeast S-phase DNA damage checkpoint will provide an important framework for understanding how the human checkpoint maintains genomic stability. This understanding will lead to new therapeutic targets and diagnostic tools for the treatment and prevention of human cancer.